Fungi, especially those which live in symbiotic association with other organisms, are rich sources of small-molecule natural products representing novel structures and eliciting diverse biological responses, but to date they remain underexplored. The goal of this project is to generate molecular libraries derived from a unique collection of carefully identified and curated endophytic and endolichenic fungal strains from four diverse U.S. eco-zones. In Specific Aim 1, selected strains of fungal species which have never been investigated previously for their secondary metabolites will be cultured in small-scale and subjected to HPLC analysis, LC-MS and 13C-NMR dereplication to select high-yielding strains producing new, rare, and/or carbonyl-containing compounds. In Aim 2, promising fungal strains will be cultured on large-scale and processed to isolate natural products new to the NIH Molecular Libraries Small-Molecule Repository (MLSMR). Aim 3 will focus on an innovative approach of chemically diversifying CO-containing natural products prevalent among fungal secondary metabolites in a high-yielding reaction with the diamine reagent, hydrazine. This will result in analogs containing N-N moieties very rare in nature but common among drug-like combinatorial and synthetic molecules. During the course of this project compounds possessing unique structural features and occupying biologically-relevant chemical space hitherto not represented in the MLSMR will be generated and submitted to the MLSMR, which constitutes Aim 4 of this proposal. The major strengths of this application are: (i) availability of a collection of a unique, geno-typed endosymbiotic fungal strains not previously investigated for their secondary metabolites; (ii) biological relevance of structures to be generated that are poorly represented in the MLSMR; (iii) the track record of the group in microbiology and natural products chemistry, as well as (iv) the success the group has had in converting carbonyl-containing fungal metabolites into their N-N bond-containing analogs, many with unprecedented structures and potential bioactivities. Thus, the likelihood of discovering activities relevant to human diseases and/or affecting the functions of genes, signaling pathways, and disease-related biological processes will be greater within these libraries due to their natural product origin and/or drug-like structures. PUBLIC HEALTH RELEVANCE: Microbial natural products have a rich history for their utility as tools for identification of novel therapeutic targets for human diseases and as lead structures for drug development. This project proposes to generate diverse molecular libraries from endosymbiotic fungi with potential to interact with cellular targets and serve as lead molecules for drug development and therefore will have a great impact on public health.